This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The proteases carboxypeptidase A and thermolysin catalyze the hydrolysis of peptide bonds. Both have a Zn in the active site bound to two histidines, one carboxylate and one solvent molecule. The binding mode of the carboxylate differs between the enzymes and also changes depending on pH (monodentate to bidentate). The attack to a metal bound hydroxide has been suggested as the first step in one of the hypothesized mechanisms. Thus, determination of the protonation state of the solvent is essential to understand the mechanism of the enzymes. XAS, due to the similar scattering phases of N from histidine and O from the solvent, cannot accurately discern the subtle difference in the metal-solvent distance due to different protonation of the solvent molecule. Moreover Zn, with its closed d-shell, does not show a pre-edge, which usually provides more features to analyze the electronic structure of the metal site. At difference, high-resolution x-ray emission measurements (XES) of the crossover peak are very sensitive to differences in protonation, as seen in former studies on carbonic anhydrase and its model compounds. In this work the crossover and Kbeta2,5 peaks of the enzymes carboxypeptidase A and thermolysin will be collected for two different pH values (about 6 and 8.5). Using molecular modeling, DFT XANES simulations and also real space multiple scattering theory, the densities of occupied states can be calculated and compared to the experimental data. The combined analysis would allow determining the protonation state of the solvent and the orientation of the carboxylate for the different values of the pH, and provide a solid base to discuss the hypothesis on the mechanisms of the enzymes.